hnRNP R negatively regulates transcription by modulating the association of P‐TEFb with 7SK and BRD4

Abstract The P‐TEFb complex promotes transcription elongation by releasing paused RNA polymerase II. P‐TEFb itself is known to be inactivated through binding to the non‐coding RNA 7SK but there is only limited information about mechanisms regulating their association. Here, we show that cells deficient in the RNA‐binding protein hnRNP R, a known 7SK interactor, exhibit increased transcription due to phosphorylation of RNA polymerase II. Intriguingly, loss of hnRNP R promotes the release of P‐TEFb from 7SK, accompanied by enhanced hnRNP A1 binding to 7SK. Additionally, we found that hnRNP R interacts with BRD4, and that hnRNP R depletion increases BRD4 binding to the P‐TEFb component CDK9. Finally, CDK9 is stabilized upon loss of hnRNP R and its association with Cyclin K is enhanced. Together, our results indicate that hnRNP R negatively regulates transcription by modulating the activity and stability of the P‐TEFb complex, exemplifying the multimodal regulation of P‐TEFb by an RNA‐binding protein.


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Appendix Figure   P l a t e 1 P l a t e 2 P l a t e 3 P l a t e 4 P l a t e 1 P l a t e 2 P l a t e 3 P l a t e 4  Appendix Figure S1. Generation of hnRNP R knockout cells

A.
Agarose gel electrophoresis of the PCR products obtained from control HeLa cells cotransfected with pCMV-PE2 and empty pU6-pegRNA-GG-acceptor, and from HeLa cells co-transfected with pCMV-PE2 and pU6-pegRNA-GG-acceptor harbouring a pegRNA targeting HNRNPR exon 4. For PCR, the common primers (red) EXON4_F and EXON4_R annealing upstream and downstream of the prime editing target region, or the primers EXON4_ AGTGA_F (purple), which recognizes the inserted nucleotides, and EXON4_R were used. Thick red arrow indicates the knockout-specific PCR product.

B.
Percentage of edited and unedited clonal colonies from four individual 96 well plates.

C.
Percentage of heterozygous and homozygous clonal colonies among edited colonies on four individual 96 well plates.

Detection of nascent RNA in hnRNP R-deficient cells revealed by 5-ethynyl-uridine (EU) labelling
A. Control reactions of HeLa cells exposed to either 5-EU or Cy3-Azide. Cells were treated with DMSO or actinomycin D as indicated. Scale bars: 10 µm.

Appendix Figure S6. Association of CDK9 with HSP70 and 90
A.
Western blot analysis of HSP90, HSP70, CDK9 and α-Tubulin in the input lysates used for co-immunoprecipitation.

B.
Western blot analysis of HSP90 and HSP70 co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.

D.
Quantification of HSP70 and 90 in the input (A). Data are mean with SD; **P ≤ 0.01; unpaired two-tailed t-test (n = 3 biological replicates). In Rabbit_IgG Anti_hnRNP R C Appendix Figure S7. hnRNP R and hnRNP A1 do not associate with P-TEFb and HEXIM1 A.
Western blot analysis of Cyclin T1, HEXIM1 and CDK9 co-immunoprecipitated by an anti-hnRNP R antibody. Immunoprecipitation with rabbit-IgG antibody was used as control.
B. Western blot analysis of HEXIM1, CDK9, hnRNP R and hnRNP A1 coimmunoprecipitated by an anti-Cyclin T1 antibody. Immunoprecipitation with mouse-IgG antibody was used as control.
C. Western blot analysis of Cyclin T1, hnRNP R and hnRNP A1 co-immunoprecipitated by an anti-CDK9 antibody. Immunoprecipitation with mouse-IgG antibody was used as control. Schematic of the strategy for the generation of 7SK knockout cells by prime editing. The T(5) sequence inserted by prime editing is marked in red, the poly(T) sequence acting as transcriptional terminator for RNA polymerase III is indicated by asterisks.
F. Quantification of relative expression of 7SK RNA by qPCR. Data are mean with SD; ***P ≤ 0.001, n.s. not significant; one-way ANOVA with Tukey's multiple comparisons test (n = 3 biological replicates).